scholarly journals Attenuation of drug-stimulated topoisomerase II–DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα

1998 ◽  
Vol 336 (3) ◽  
pp. 727-733 ◽  
Author(s):  
Masako AOYAMA ◽  
Dale R. GRABOWSKI ◽  
George R. DUBYAK ◽  
Andreas I. CONSTANTINOU ◽  
Lisa A. RYBICKI ◽  
...  
Keyword(s):  
Dna Damage ◽  
Complex Formation ◽  
Topoisomerase Ii ◽  
Wild Type ◽  
Acetoxymethyl Ester ◽  
Free System ◽  
Site Specific ◽  
Calcium Buffer ◽  
Topo Ii ◽  
Cleavable Complex

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIα phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II–DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II–DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N´,N´-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II–DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIα. Tryptic phosphopeptide mapping of topo IIα protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746–29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIα are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

scholarly journals Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway

2004 ◽  
Vol 166 (4) ◽  
pp. 517-526 ◽  
Author(s):  
Alexei Mikhailov ◽  
Mio Shinohara ◽  
Conly L. Rieder
Keyword(s):  
Dna Damage ◽  
Histone Deacetylase ◽  
P38 Mapk ◽  
Topoisomerase Ii ◽  
Histone Deacetylase Inhibitors ◽  
Chromatin Modification ◽  
Early Prophase ◽  
Global Changes ◽  
Atm Kinase ◽  
Topo Ii

When early prophase PtK1 or Indian muntjac cells are exposed to topoisomerase II (topo II) inhibitors that induce little if any DNA damage, they are delayed from entering mitosis. We show that this delay is overridden by inhibiting the p38, but not the ATM, kinase. Treating early prophase cells with hyperosmotic medium or a histone deacetylase inhibitor similarly delays entry into mitosis, and this delay can also be prevented by inhibiting p38. Together, these results reveal that agents or stresses that induce global changes in chromatin topology during G2 delay entry into mitosis, independent of the ATM-mediated DNA damage checkpoint, by activating the p38 MAPK checkpoint. The presence of this pathway obviates the necessity of postulating the existence of multiple “chromatin modification” checkpoints during G2. Lastly, cells that enter mitosis in the presence of topo II inhibitors form metaphase spindles that are delayed in entering anaphase via the spindle assembly, and not the p38, checkpoint.

scholarly journals Uncoupling DNA damage from chromatin damage to detoxify doxorubicin

2020 ◽  
Vol 117 (26) ◽  
pp. 15182-15192 ◽  
Author(s):  
Xiaohang Qiao ◽  
Sabina Y. van der Zanden ◽  
Dennis P. A. Wander ◽  
Daniel M. Borràs ◽  
Ji-Ying Song ◽  
...  
Keyword(s):  
Dna Damage ◽  
Side Effects ◽  
Topoisomerase Ii ◽  
Prolonged Treatment ◽  
Anticancer Efficacy ◽  
Strand Breaks ◽  
Human Acute Myeloid Leukemia ◽  
Topo Ii ◽  
Dose Dependent

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.

Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis

1994 ◽  
Vol 107 (10) ◽  
pp. 2887-2898
Author(s):  
M. Morse-Gaudio ◽  
M.S. Risley
Keyword(s):  
Dna Damage ◽  
Xenopus Laevis ◽  
Topoisomerase Ii ◽  
Spermatogenic Cell ◽  
Spermatogenic Cells ◽  
Dna Breaks ◽  
Whole Cells ◽  
Topo Ii ◽  
Pachytene Spermatocytes ◽  
Antipeptide Antibody

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.

Casein Kinase II Modulates Topoisomerase II Alpha Nuclear Export and Drug Sensitivity in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4032-4032
Author(s):  
Joel G Turner ◽  
Jana L Dawson ◽  
Daniel M Sullivan
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Nuclear Export ◽  
Topoisomerase Ii ◽  
Myeloma Cell ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Topoisomerase Ii Alpha ◽  
Myeloma Cells ◽  
Topo Ii

Abstract Abstract 4032 Nuclear export of topoisomerase II alpha (topo IIα) to the cytoplasm results in de novo resistance to topo IIα inhibitors in hematological malignancies. We have previously demonstrated that topo IIα is exported from the nucleus of human multiple myeloma cells by a CRM1-dependent mechanism at densities similar to those in myeloma patient bone marrow (Engel et al, Exp. Cell Res. 295:421-31, 2004). We have also identified the nuclear export signals for topo IIα at amino acids 1017–1028 and 1054–1066 using mutated full-length FLAG-topo IIα protein and immunofluorescence microscopy, (Turner et al, J. Cell Sci. 117:3061-71, 2004). In addition, blocking nuclear export with a CRM1 inhibitor or by using CRM1-siRNA sensitizes myeloma cells to topo II poisons (Turner et al. Cancer Res. 69:6899-905, 2009). Therefore, preventing nuclear export may sensitize myeloma cells to topo II inhibitors. To determine what signals CRM1-mediated nuclear export of topo IIα we investigated the phosphorylation status of topo IIα, both in the nucleus and in the cytoplasm, using proteomic technologies. Topo IIα was isolated from the nuclei and cytoplasm of human myeloma cells by immunoprecipitation. Purified topo IIα fractions were analyzed by Orbitrap mass spectroscopy. We found that serine 1524 was highly phosphorylated in the cytoplasmic fraction. Using site-directed mutagenesis we converted serine 1524 to an alanine to determine if mutated FLAG-tagged topo IIα export was reduced as compared to wild-type FLAG-tagged topo IIα. Serine 1524 is a CK2 phosphorylation site, therefore, we tested a recently published and highly specific inhibitor of CK2; CX-4945 (Ferguson et al, FEBS Letters 585:104–110, 2011). This drug was used in combination with the topo IIα inhibitor doxorubicin and assayed for apoptosis by caspase 3 cleavage and flow cytometry both in vitro using myeloma cell lines and ex vivo using patient bone marrow mononuclear cells. Using mass spectroscopy we found that cytoplasmic but not nuclear topo IIα was phosphorylated at serine 1524, a published CK2 motif. Using site-directed mutagenesis we converted serine 1524 to an alanine and found that mutated FLAG-topo IIα export was reduced, as compared to wild-type FLAG- topo IIα. The CK2 inhibitor CX-4945 induced apoptosis in both myeloma cell lines and in myeloma patient bone marrow aspirates. In addition, we found that CX-4945 prevented nuclear export of topo IIα in high-density cells. These data were duplicated using a CK2 specific siRNA to knockdown CK2 expression (90% knockdown). Blocking nuclear export of topo IIα with the CK2 inhibitor CX-4945 or CK2-specific siRNA sensitized drug-resistant myeloma cells to the topo II poison doxorubicin. These data may have potential clinical implications in the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cleavable-Complex Formation by Wild-Type and Quinolone-Resistant Streptococcus pneumoniae Type II Topoisomerases Mediated by Gemifloxacin and Other Fluoroquinolones

2002 ◽  
Vol 46 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Genoveva Yague ◽  
Julia E. Morris ◽  
Xiao-Su Pan ◽  
Katherine A. Gould ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Complex Formation ◽  
Dna Cleavage ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Drug Induced ◽  
Drug Concentrations ◽  
Cleavable Complex

ABSTRACT Gemifloxacin is a recently developed fluoroquinolone with potent activity against Streptococcus pneumoniae. We show that the drug is more active than moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin against S. pneumoniae strain 7785 (MICs, 0.03 to 0.06 μg/ml versus 0.25, 0.25, 1, and 1 to 2 μg/ml, respectively) and against isogenic quinolone-resistant gyrA-parC mutants (MICs, 0.5 to 1 μg/ml versus 2 to 4, 2 to 4, 16 to 32, and 64 μg/ml, respectively). Gemifloxacin was also the most potent agent against purified S. pneumoniae DNA gyrase and topoisomerase IV in both catalytic inhibition and DNA cleavage assays. The drug concentrations that inhibited DNA supercoiling or DNA decatenation by 50% (IC50s) were 5 to 10 and 2.5 to 5.0 μM, respectively. Ciprofloxacin and levofloxacin were some four- to eightfold less active against either enzyme; moxifloxacin and gatifloxacin showed intermediate activities. In assays of drug-mediated DNA cleavage by gyrase and topoisomerase IV, the same order of potency was seen: gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin ≈ ciprofloxacin. For gemifloxacin, the drug concentrations that caused 25% linearization of the input DNA by gyrase and topoisomerase IV were 2.5 and 0.1 to 0.3 μM, respectively; these values were 4-fold and 8- to 25-fold lower than those for moxifloxacin, respectively. Each drug induced DNA cleavage by gyrase at the same spectrum of sites but with different patterns of intensity. Finally, for enzymes reconstituted with quinolone-resistant GyrA S81F or ParC S79F subunits, although cleavable-complex formation was reduced by at least 8- to 16-fold for all the quinolones tested, gemifloxacin was the most effective; e.g., it was 4- to 16-fold more active than the other drugs against toposiomerase IV with the ParC S79F mutation. It appears that the greater potency of gemifloxacin against both wild-type and quinolone-resistant S. pneumoniae strains arises from enhanced stabilization of gyrase and topoisomerase IV complexes on DNA.

scholarly journals DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

1997 ◽  
Vol 17 (7) ◽  
pp. 4070-4079 ◽  
Author(s):  
M Stanulla ◽  
J Wang ◽  
D S Chervinsky ◽  
S Thandla ◽  
P D Aplan
Keyword(s):  
Dna Cleavage ◽  
Cellular Response ◽  
Topoisomerase Ii ◽  
Apoptotic Cell ◽  
Chemotherapeutic Agents ◽  
Higher Order ◽  
Site Specific ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Topo Ii

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.

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